Identifying Androsterone (ADT) as a Cognate Substrate for Human Dehydroepiandrosterone Sulfotransferase (DHEA-ST) Important for Steroid Homeostasis

Abstract

In steroid biosynthesis, human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3-phosphoadenosine-5-phosphosulfate to dehydroepiandrosterone (DHEA). DHEA and its sulfate play roles as steroid precursors; however, the role of the enzyme in the catabolism of androgens is poorly understood. Androsterone sulfate is clinically recognized as one of the major androgen metabolites found in urine. Here it is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA. The structure of human DHEA-ST in complex with ADT has been solved at 2.7 Å resolution, confirming ADT recognition. Structural analysis has revealed the binding mode of ADT differs from that of DHEA, despite the similarity of the overall structure between the ADT and the DHEA binary complexes. Our results identify that this human enzyme is an ADT sulfotransferase as well as a DHEA sulfotransferase, implying an important role in steroid homeostasis for the adrenals and liver.Sulfonation is catalyzed by a family of sulfotransferases that conjugate a sulfonate group (SO 3 ) from 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) 1 to a hydroxyl group of the recipient molecule. With desulfation by sulfatases, sulfonation has been considered as one of the major enzymatic reactions in the metabolism not only of endogenous compounds and xenobiotics, but also of steroid hormones. In most cases, the transfer of the charged sulfonate moiety to an acceptor steroid decreases the biological activity of the steroid. Indeed, steroid sulfates resulting from this reaction are not capable of binding to or activating steroid receptors. In addition, the sulfonation reaction increases water solubility of steroids and thereby enhances their excretion into the urine and/or bile (1, 2).Human dehydroepiandrosterone sulfotransferase (DHEA-ST; SULT2A1; EC 2.8.2.2) was identified mainly from human liver and adrenals, using Northern blot analysis (3) and RT-PCR analysis (4). A single isoform of DHEA-ST from human liver and adrenal tissues was confirmed by the expression and purification of the enzyme from these organs (5, 6), molecular cloning studies (7) and the comparison study of the physical, kinetic, and immunological properties of liver and adrenal forms of the enzyme (8). Steroid sulfonation has been recognized as an important means for maintaining steroid hormone levels in their metabolism. In humans, dehydroepiandrosterone sulfate (DHEAS) is the most prodigious steroid precursor and one of the major secretory products of both adult and fetal adrenals. In the fetoplacental-maternal unit (the unique interdependence of fetus, placenta, and mother) shown in Scheme 1, DHEAS plays an important role as the major precursor for placental estrogen biosynthesis, thus maintaining pregnancy. A considerable amount of DHEAS is mainly produced from the fetal zone in the adrenal gland (9). Then DHEAS...

Identification and Characterization of Two Amino Acids Critical for the Substrate Inhibition of Human Dehydroepiandrosterone Sulfotransferase (SULT2A1)

“…The structures of the M137I and M137W were determined by molecular replacement as implemented in CNS v1.1 (Brunger et al, 1998) using the crystal structure of human dehydroepiandrosterone sulfotransferase (Protein Data Bank code 1OV4) (Chang et al, 2004) as a search model. The M137I and M137W molecule was located in the asymmetry unit after rotation and translation function searches.…”

“…The catalysis and inhibition of DHEA and ADT by SULT2A1 have been reported to regulate the homeostasis and metabolism of these compounds and to maintain steroid levels (Chang et al, 2004). Previous studies have suggested that the onset of substrate inhibition is the formation of ternary dead-end complex (Duffel and Jakoby, 1981;Zhang et al, 1998).…”

“…Structural alignments among the complex structures of SULT2A1/ADT, SULT2A1/DHEA, and SULT2A1/PAP (Pedersen et al, 2000;Rehse et al, 2002;Chang et al, 2004) reveal spatial variation of amino acids in different substrate and PAP binding states. We herein describe experimental considerations that lead us to propose that two of the amino acids, Tyr-238 and Met-137, are residues responsible in regulating substrate inhibition.…”

“…Steroid sulfation has been recognized as an important process for maintaining steroid hormone levels during their metabolism. In humans, dehydroepiandrosterone sulfate is the most prevalent steroid precursor and is one of the major secretory products of both adult and fetal adrenals (Chang et al, 2004).…”

Unique potentiometric detection systems for HPLC determination of some steroids in human urine

“…The latter makes us conclude that potentiometry as a detection technique is more suitable than UV -Vis spectrophotometry for these GP derivatized 17-KS compounds, i.e., cationic ketosteroid pyridinium acetohydrazones. The underlying reason for not detecting the naturally occurring 17-KS concentrations in used human urine samples is that in such natural urine samples they are secreted mainly in their conjugated sulfated-and/or glucuronidated forms [7,48].…”